The objective of this proposal is to develop a technologyfor genome sequencing. In the R21 phase of the project, we propose to develop a method for fabricating high-density arrays of wells with sub-micrometer dimensions for ordering single nanoparticles and DNA molecules. We will demonstrate the proof-of-principle of a new method for DNA sequencing called single molecule sequencing by ligation (SM-SBL). More than one billion individual DNA molecules can be sequenced in massive parallel by cyclic sequencing by ligation using photocleavable reversible oligonucleotide probes. Since both ends of a template can sequenced with SM-SBL, high genome coverage with paired-end information can be obtained. We proposeto investigate the feasibility of de novo genome sequencing using this powerful technology, both experimentally and computationally. In the R33 phase of the project,we propose to improve the SM-SBL method even further by developing a strategy for encoding the 64 oligonucleotide probeswith fluorescence nanoparticles for reading 3 bases per sequencing cycle. The nanoparticles will be encoded with a combination of multiple wavelengths and intensity levels by embedding the fluorescent molecules in different molar ratios into the nanoparticles. A new method for high throughput fluorescenceimaging is also proposed. A quad-bandpass filter and beamsplitter will be combined with a light source capable of ultra-fast wavelength selection for fluorescence imaging. Microfabrication techniques will be used to order the target molecules, to build in sign posts and to match the spacing of the molecules to the optical imaging system allowing for maximum data throughput. A highly integrated system for genome-scalesequencing will be developed. Algorithms and software for genome sequence assembly from paired-end sequencedata will also be developed. The novel technology will enable DNA sequencing from paired-ends of single DNA molecules in an extremely massive parallel process, allowing rapid and low-costde novo genome sequencing. Once developed, this kind of technology will enable routine sequencing of individual human genomes, revolutionizing biomedical research and healthcare.